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OnSiteGene Developing Ultra-Fast qPCR System for Rapid Syndromic Testing

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NEW YORK (GenomeWeb) – San Diego-based startup OnSiteGene is developing an ultra-fast molecular diagnostics platform that can perform highly multiplexed real-time PCR assays in about five minutes. The firm plans to build on the throughput and multiplexing capabilities of its instrument to develop customizable syndromic infectious disease panels, allowing users to select which molecular tests to run for each patient sample.

The firm is developing assays for meningitis as well as an influenza A and B test, but it expects its first commercial test to be a respiratory panel of six targets.

OnSiteGene showed a prototype instrument at the American Association for Clinical Chemistry annual meeting in Chicago earlier this month. Called Peak V, it is currently a benchtop instrument, but OnSiteGene Chairman Thomas Gong said that the company is working to develop it into a smaller platform that could be used in near-patient settings for "on the spot" molecular diagnostic evaluations.

Decreasing the speed of diagnostic testing is an important mission of the company, Gong said. He noted that the work of Carl Wittwer and associates has been seminal in this regard, since Wittwer proved the feasibility of ultra-fast PCR with a method called Extreme PCR that decreases the speed of the reaction, in part by increasing the concentration of primers and polymerases, as previously reported.

Elements of Wittwer's early work were incorporated into the LightCycler instrument from Idaho Technology, which later changed its name to BioFire Diagnostics. Roche licensed the LightCycler technology from then-Idaho Tech and further developed it into its flagship PCR system.

More recently, an instrument called NextGen PCR manufactured by Dutch firm Molecular Biology Systems and distributed in the US and Canada by Canon BioMedical runs super-fast PCR, while instruments from Thermal Gradient can perform qPCR. The former claims two-minute, 30-cycle PCR amplification of 100-base-pair fragments, while the latter claims run times between two and eight minutes for a 40-cycle qPCR.

The current version of the MBS system uses 96- and 384-well microplates that are sealed with polypropylene foil, with wells holding between five and 20 microliters, proprietary foil-piercing pipette tips to recover PCR products from the sealed microplates, and spatial thermal cycling, in which sample is shuttled between heating and cooling elements. The Thermal Gradient system also employs spatial thermal cycling, as well as microfluidic test cartridges.

The OnSiteGene system, on the other hand, is being designed to handle standard PCR tubes, and it uses spatial thermal cycling, but with a twist.

In general, temporal PCR takes about one hour because heating and cooling is a slow process when a PCR tube sits in the well of a heat block, Gong said. The temperature of the block must first change, followed by the temperature of the tube, he said. Spatial thermal cycling systems can be designed that use robotic arms and plunge sample into heating and cooling sources, but a solid heat source has issues with contact between the source and the tube, while using hot and cold air is not ideal because air is not a good heat conductor, Gong said. Water was a medium of choice in old-school PCR systems, but it is messy and can easily become contaminated.

Gong said the OnSiteGene team reasoned that liquid metal would be the ideal medium, and then set out to find an alloy that could suit its needs. The result is proprietary, he said, but is able to take standard volumes of sample in 0.2-milliliter PCR tubes from 60 °C to 95 °C in about 1.5 seconds. The platform also accepts sample in standard glass capillary tubes, which are placed in a disposable plastic cartridge for safety.

"Typically, a glass capillary tube can achieve three- to six-minute 40-cycle real-time PCR, and the 0.2-ml plastic PCR tube can achieve five- to nine-minute 40-cycle real-time PCR," Gong said.

The firm has reasoned that a typical general practitioner sees 40 patients per day, and half of these have complaints involving infectious diseases, so Gong calculates that eight minutes is the maximum one would want a test to take in order to run and report 20 per day.

OnSiteGene's technology has also built on and expanded the Extreme PCR concept, showing that standard concentrations of reagents can be used to attain extension and annealing as well. Gong noted that this makes the system more feasible in terms of manufacturing, bringing it to a cost level on par with standard lab-based qPCR. Furthermore, to determine if enhanced speed might sacrifice sensitivity, OnSiteGene has compared the system to a Thermo Fisher Scientific QuantStudio real-time PCR machine and found equivalent detection down to the lowest serial dilutions of template, Gong said, adding that the Peak V achieved these results in five minutes compared to one hour.

Peak V is also able to carry out sample preparation to extract and purify nucleic acids from raw samples like blood and nasal swab, Gong said, to achieve fully-automated sample-in, answer-out test operation.

Importantly, the system can also be used for multiplexing, with four optical channels per tube covering a broad range of detection channels.

Thus, unlike current commercially-available rapid near-patient and point-of-care molecular diagnostics platforms – such as the Roche Liat, Abbott Alere i, Cepheid GeneXpert Xpress, and Mesa Accula – Gong said the OnSiteGene machine is different because it can potentially run syndromic panels.

BioMérieux subsidiary BioFire makes a near-patient, CLIA-waived version of its FilmArray Respiratory Panel EZ that detects 11 viruses and three bacterial pathogens from nasopharyngeal swabs with two minutes of hands-on time, but the throughput of that system is one test per hour.

Gong said each of the 32 reactors in the Peak V can support a multiplex test with up to four targets. The firm is now focusing on developing an "editable panel," Gong said, that will essentially be a series of assays or smaller multiplex syndromic panels, each hopefully approved by the US Food and Drug Administration, such that users can run any combination. By contrast, although they have been adopted rapidly by clinical labs, commercial syndromic panels tend to be single assays that can't be broken up, which Gong noted can be problematic if targets are not epidemiologically relevant, and can also pose issues in terms of reporting results that weren't expressly ordered by physicians.

With Peak V's four optical channels and 32 glass capillary reactors, if a 4-plex PCR is conducted in each reactor, the system can theoretically test one sample against 128 targets simultaneously, Gong said.

"This large multiplexing can be reduced if more samples are tested, for example if we test eight samples at a time, each sample can be tested against a panel of 16 targets," he added.

By contrast, the non-waived BioFire panels typically run around two dozen targets, for example, while a platform from Qiagen subsidiary Stat-Dx can run panels with up to 48 targets. A system from Curetis, meanwhile, can run around 100 targets from a single patient sample using endpoint PCR coupled with array-based detection.

OnsiteGene is currently focusing on developing a respiratory panel on a configuration of Peak V that has integrated sample preparation, Gong said, and in the long term the company aims to become a pioneer in superfast syndromic panel testing, bring syndromic panel testing to sites requiring immediate test results.

"We wish to collaborate with academic and research institutions, testing labs, hospitals, and assay development companies to bring superfast qPCR sample-in, answer-out testing to various markets," he said.

OnSiteGene is currently collaborating with an outside institute for a case study on a meningitis molecular diagnostic, Gong said, which he anticipates will be published in the next six months or so.

It has started validation of a six-target respiratory panel test and begun the process of getting guidance from the FDA, he said, estimating that it will take about two years for the firm to obtain 510(k) clearance on a first product and the instrument.

In the meantime, the company is in the process of redesigning the Peak V instrument. Gong said the firm is planning to reduce its size, although not to the level of a hand-held system such as the recently cleared Mesa Biotech flu test. The goal is to get the instrument to a where it can but put on a table in a physician's office or in an emergency room, Gong said.

OnSiteGene is privately-held and has about 60 employees. It has some shareholders in common with the Star Array group from Singapore, Gong said. The firm has raised about $10 million from private investors in the US and Singapore since 2015, and is planning a fundraising round in the middle of 2019.

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